In the process of the growth of seed plants, embryos in seeds develop so as to form cotyledons and apical meristems (shoot apical meristems). Cell division of the apical meristem (shoot apical meristem) causes leaf primordia to be sequentially formed, and causes axillary meristems to be formed on an adaxial side of the leaf primordia. The axillary meristems then serve as apical meristems (shoot apical meristems) and result in axillary buds. During vegetative growth of a plant, usually, the development of axillary buds is temporarily in a dormant state (suppressed). In a case where apical meristems (shoot apical meristems) of a primary shoot is transitioned from a vegetative growth state to a reproductive growth state, or in a case where the apical meristems (shoot apical meristems) die, the development of the axillary buds is no longer in a dormant state and is promoted. With respect to the development of axillary buds, there are a plurality of research reports on solanaceous plants (e.g., tomatoes and tobaccos) and on other plants (e.g., rice and Arabidopsis thaliana).
A tobacco plant, which is cultivated for harvesting leaves, is subjected to topping (cutting off a stem of an apical portion with a flower) during cultivation, for the purpose of enhancing the quality and quantity of leaves to be harvested (e.g., for the purpose of accumulating composition of the leaves and maturing and expanding leaves). Topping causes axillary buds of the tobacco plant to start vigorously developing from, bases of leaves (leaf axil). The development of axillary buds naturally consumes nutrients, and therefore causes a relative decrease in nutrient which are supplied to leaves to be harvested. Therefore, the development and outgrowth of axillary buds leads to a decrease in quality and yield of leaves to be harvested. For a reason similar to that for topping, axillary buds are subjected to a treatment, such as removal or developmental suppression, during a period between topping and harvesting of leaves. Note that in the case of at least tobacco plants, it is known that even after an axillary bud is removed, axillary buds repeatedly develop from a base of the same leaf. Therefore, in cultivation of tobacco plants for harvesting leaf tobaccos, control of axillary buds is an important issue that should be dissolved.
Examples of a method of removing an axillary bud encompass a method in which an axillary bud is picked by hand or by machine. Picking an axillary bud by hand involves (i) a large amount of work (and accordingly an increase in labor costs) and (ii) a problem of low efficiency. Picking an axillary bud by machine is less accurate than picking by hand, and therefore brings a problem of damaging a plant. Examples of a method of suppressing the development of an axillary bud encompass (i) suppression by use of agrochemicals and (ii) suppression by genetic modification. The use of agrochemicals involves problems such as repeated application for maintaining an effect, an impact on the growth of a plant, an impact on leaves to be harvested due to agrochemicals residue, and an increase in inspection cost for agrochemicals residue.
Note that Patent Literatures 1 and 2 and Non-Patent Literatures 1 through 19 disclose matters in regard to development of axillary buds. Patent Literatures 1 and 2 disclose techniques for suppressing the development of axillary buds.
With reference to Non-Patent Literatures 1 through 19, genes involved in the formation of axillary meristem will be described below.
A plurality of genes from plants other than tobacco plants have been reported as genes involved in the formation of axillary meristem. Representative examples of such a gene encompass LATERAL SUPPLESSOR (LS), Blind (B1), REVOLUTA (REV), and CUP-SHAPED COTYLEDON (CUC).
It has been reported that LS is isolated from Arabidopsis thaliana (Non-Patent Literature 1), tomato (Non-Patent Literature 2), and rice (Non-Patent Literature 3), and is a gene necessary for the formation of an axillary meristem. In a mutant of LS gene of Arabidopsis thaliana, while axillary buds of cauline leaves were normal, axillary buds of rosette leaves other than two topmost rosette leaves were hardly observed (Non-Patent Literature 1). In a mutant of LS gene of a tomato, while axillary buds during a vegetative stage were not present, axillary buds were formed at two topmost parts during a reproductive stage (Non-Patent Literature 2). In a mutant of LS gene of rice (moc1), no tillers, which are equivalent to axillary buds of rice, were observed at all during both a tillering stage and a heading stage (Non-Patent Literature 3). Regarding tobaccos, while the cDNA sequence predicted as an LS orthologue gene is published (Accession number: EU0935581.1), the function of the gene in tobaccos is not confirmed.
B1 gene is isolated from Arabidopsis thaliana (Non-Patent Literatures 4 and 5) and tomato (Non-Patent Literature 6). In tomatoes, even in a case where topping had been performed, axillary buds were hardly formed regardless of leaf position, due to a mutant of one gene (Non-Patent Literatures 6 and 7). Regarding Arabidopsis thaliana, at least three genes which are redundant and B1 orthologue (REGULATOR OF AXILLARY MERISTEM (RAX) 1, 2, and 3) have been reported. While RAX1 single mutant showed suppression of axillary buds, in RAX1, 2, 3 triple mutants, axillary buds of rosette leaves were hardly formed and those of cauline leaves were largely reduced (Non-Patent Literatures 4 and 5). In the RAX1 single mutants, even after topping, the outgrowth of axillary buds from bottom rosette leaves where no formation of axillary buds was observed before topping was not observed. Based on homology comparison between (i) the putative amino acid sequences predicted from the RAX gene sequence of Arabidopsis thaliana and (ii) the putative amino acid sequence predicted from genome sequences of grape and tomato, it was predicted that tomato orthologous genes of RAX1 of Arabidopsis thaliana include C gene other than Blind. However, the C gene was not relevant to the formation of axillary buds, but was relevant to morphogenesis of leaves (Non-Patent Literature 8). Although there has not been any report on a cDNA sequence predicted as B1 orthologue gene in tobaccos, putative amino acid sequence predicted from an EST sequence identical by 93% to the amino acid sequence of tomato B1 has been published (Accession number: FS402940.1). However, the function of the gene in tobacco remains unknown.
REV gene was isolated from Arabidopsis thaliana (Non-Patent Literatures 10 and 11). In a mutant of REV, the formation of axillary buds was decreased at both rosette leaves and cauline leaves, and promotion of the formation of an axillary meristem by decapitation was not observed (Non-Patent Literatures 9, 10, and 12). Although there has not been any report on a cDNA sequence predicted as REV orthologue gene in tobaccos, putative amino acid sequence predicted from an EST sequence identical by 79% on an amino acid level to Arabidopsis thaliana REV has been published (Accession number: FG135778.1). In addition, a full-length cDNA sequence predicted as REV orthologous gene in a tobacco (variety: SR-1) has been published (Accession number: JQ686937). However, there has not been any report on the function of a gene, in a tobacco, which is highly homologous to the REV.
Three genes (CUC1, CUC2, and CUC3) as CUC are isolated from Arabidopsis thaliana (Non-Patent Literatures 16 through 18). The function of both CUC1 and CUC2 is control of shoot apical meristems and redundant (Non-Patent Literature 15). Although cuc3 single mutation repressed formation of axillary buds, cuc2 and 3 double mutation showed enhanced repression (Non-Patent Literatures 13 and 14). Although there has not been any report on a cDNA sequence predicted as CUC orthologue in tobaccos, putative amino acid sequence predicted from an EST sequence (FG644078.1) identical by 81% to the amino acid sequence of NAM domain sequence, which is a conserved domain of CUC1 gene of Arabidopsis thaliana, has been published. It has also been reported that RNAi transgenic tobacco using the sequence predicted as CUC3 of Apocynum venetum showed reduced expression of a certain gene (the sequence is not published) and morphological abnormality of leaves shown in CUC mutants of Arabidopsis thaliana (Non-Patent Literature 19). However, the function of a gene, in a tobacco, which gene is highly homologous to CUC, is not clear, and, at least, the function with respect to an axillary bud has not been reported.